Human metapneumovirus (HMPV) was first reported in the Netherlands in 2001 and soon after was isolated in patients with respiratory tract disease throughout the world, particularly in the pediatric population. HMPV replicates inefficiently in cell culture, posing a challenge to research. The contribution of HMPV to human disease remains to be defined, but it appears to account for approximately 5 to 15% of pediatric hospitalizations due to respiratory tract disease. We are using reverse genetic methods to develop attenuated strains of HMPV for use as a live intranasal pediatric vaccine, one that would be given in combination with live vaccines currently being developed by LID/NIAID against human respiratory syncytial virus (HRSV) and the human parainfluenza viruses (HPIVs). HMPV is an enveloped virus with a genome that is a single negative-sense strand of RNA. It is classified in the paramyxovirus family together with HRSV and the HPIVs. We recently described the first complete sequence of the HMPV genome, and prepared complete consensus sequences for viruses (CAN97-83 and CAN97-75) representing the two genetic subgroups of HMPV (A and B, respectively). The HMPV genomes sequenced to date range in length from 13,280-13,335 nt. The genome contains 8 genes that are in the order 3?-N-P-M-F-M2-SH-G-L-5? and have open reading frames corresponding to 9 major proteins. By analogy to HRSV, which has been studied in much greater detail, the HMPV proteins are: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; M2-1, RNA synthesis factor; M2-2, RNA synthesis factor; SH, small hydrophobic protein of unknown function; G, attachment glycoprotein; and L, viral polymerase. The two HMPV subgroups share 81% nucleotide identity and 88% aggregate amino acid identity, similar to the respective values of 81% and 88% for the two HRSV subgroups. We developed a reverse genetic system for the CAN97-83 isolate, whereby complete infectious virus can be generated entirely from cloned cDNAs transfected into cultured cells. This provides a method for introducing predetermined changes into infectious HMPV for the purpose of basic molecular genetic studies and for designing vaccines. In one study, we designed a version of HMPV, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette placed 58 nt from the 3' end of the genome. This helped define the viral cis-acting signals necessary to transcribe a gene into mRNA. This virus is being used to monitor viral infection directly in living cells. In addition, we used the HMPV-GFP virus to develop a more rapid and reliable assay for detecting HMPV-neutralizing antibodies. In another study, HMPV was engineered to delete the SH and G genes in their entirely individually and in combination. The del-SH, del-G, and del-SH/G deletion mutants were readily recovered and were found to replicate in vitro with an efficiency that was approximately equivalent to that of wild type virus. This showed that the SH and G proteins are not essential for efficient growth in cell culture. The SH, G and F proteins were identified for the first time by immunoprecipitation using peptide-specific sera. This showed that the SH protein accumulates in a variety of forms that range in apparent electrophoretic mobility from 23-220 kDa, with the differences appearing to be due to glycosylation. The G protein also appeared to be heavily glycosylated. When administered intranasally to hamsters, the del-SH virus replicated at least as efficiently as wild-type rHMPV. This indicates that SH is completely dispensable in vivo and that its deletion does not confer a significant attenuating effect, at least in this rodent model. The del-G and del-SH/G mutants also replicated in both the upper and lower respiratory tract, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were found to be strongly attenuated for replication in both the upper and lower respiratory tract (at least 600-fold and 40-fold reduction, respectively, of mean titer on day 3 post infection compared to wild-type rHMPV). The immunogenicity of the del-SH virus was comparable to that wild-type rHMPV, consistent with its high level of replication. Although the del-G and del-SH/G viruses were strongly attenuated, they induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Thus, the del-G and del-SH/G viruses represent promising vaccine candidates that will be prepared for clinical evaluation. It is feasible to consider an HMPV vaccine virus lacking one or both of these surface proteins because other ongoing work indicates that F is the major neutralization and protective antigen whereas, somewhat surprisingly, SH and G do not appear to be significant neutralization and protective antigens. Additional mutants were made involving the M2 gene, which encodes an mRNA with two overlapping ORFs that have the potential to encode two separate proteins M2-1 and M2-2. Expression of both ORFs was confirmed. Recombinant HMPVs were generated in which expression of M2-1 and M2-2 was silenced individually or together (del-M2-1, del-M2-2, and del-M2[1+2]). Each mutant virus directed efficient multi-cycle growth in Vero cells, showing that neither protein is required for HMPV replication. The del-M2-2 virus exhibited a 3- to 9-fold increase in the accumulation of mRNA normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in the hamster model. Animals infected intranasally with del-M2-1 or del-M2(1+2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 post-infection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with del-M2-2, virus was recovered from only 1 of 12 animals, and only in the nasal turbinates on a single day. However, these animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The del-SH, del-G and del-M2-2 viruses were analyzed further in African green monkeys, an experimental animal that is anatomically and phylogenetically more closely related to humans. Del-SH replicated to a level comparable to that of the parental virus, whereas del-G was slightly attenuated in the upper respiratory tract and more than 1000-fold attenuated in the lower respiratory tract. The del-M2-2 virus was 160-fold attenuated in the upper respiratory tract, and 4000-fold attenuated in the lower respiratory tract. This confirmed that SH, G and M2-2 are nonessential accessory proteins. Induction of neutralizing antibodies by each mutant virus was efficient and comparable to wild-type HMPV. Upon challenge with wild-type HMPV, each of the three deletion mutants conferred essentially complete protection of the lower respiratory tract and complete protection (del-SH) or greater than 1000-fold reduction of challenge virus replication (del-G, del-M2-2) in the upper respiratory tract. Thus, at least two promising HMPV vaccine candidates with attenuation based on independent mutations involving the G or M2 genes are available and will be developed for phase 1 clinical testing in humans.